Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) Schematic diagram of the potential N -glycosylation sites (N X (S/T)) on the ß4 integrin subunit. The sites corresponding to the putative N -glycosylation sites on the ß4 integrin subunit (Asn 327 , Asn 491 , Asn 579 , Asn 617 , and Asn 695 ) are shown by flags. Numbers and boxes indicate the number of amino acid residue and the four intracellular fibronectin type III repeats, respectively. TM, transmembrane region. (B) Cell lysates from normal keratinocytes were immunoprecipitated using a control IgG or an anti-ß4 integrin Ab. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the biotinylated L 4 -PHA lectin (upper left panel) and E 4 -PHA lectin (upper right panel) or anti-ß4 and anti-α6 integrin Abs (lower panels). IB, immunoblot. The black and white arrowheads indicate the ß4 integrin and α6 integrin subunits, respectively. Ordinates indicate molecular sizes in kDa of marker proteins. (C) Cell morphology of the WT and ΔNß4 integrin-expressing keratinocytes during cell culture. (D) The cell-spreading area in A was calculated using computer software (Image J). Each bar represents the mean ± S.D. of triplicate assays. *p<0.001 (unpaired t-test vs.WT). (E) 20 µg of cell lysates from control lacZ- (lac), WTß4, ΔNß4 integrin-expressing keratinocytes were run on a 6% gel under reducing conditions, blotted onto a nitrocellulose membrane, and then probed with an anti-ß4 integrin Ab. (F) Cell lysates were immunoprecipitated using a polyclonal Ab against ß4 integrin. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the indicated biotinylated lectins or an anti-ß4 integrin Ab. (G) Cell surface expression levels of α3, α6, ß1 and ß4 integrin subunits of WT or ΔNß4 integrin-expressing keratinocytes were examined using FACS analysis. Prior to analysis, cells were incubated with either the indicated integrin Abs or control IgG, followed by incubation with Alexa Fluor conjugated secondary Abs, as described under “ .” (H) Immunoprecipitates from WT or ΔN keratinocytes with an anti-α6 integrin were probed with an anti-ß4 (upper panel) or ß1 integrin (lower panel) Ab.

Article Snippet: Biotinylated DSA, L 4 -PHA, E 4 -PHA, ConA, SSA and MAM lectins were from Seikagaku Biobusiness Corporation (Tokyo, Japan).

Techniques: Immunoprecipitation, Western Blot, Marker, Expressing, Cell Culture, Software, Incubation